Human cytomegalovirus (hCMV) is a widely distributed pathogen that usually establishes asymptomatic, life-long persistence in 40-80% of the human population depending on geographical and socioeconomic origin. However, in immunocompromised patients, such as transplant recipients and HIV infected individuals, and also in newborns, hCMV infection is a major cause of morbidity and mortality and puts a significant economic burden on health care systems.
hCMV is the most significant infection impacting on the outcome of solid organ transplantation (SOT) and hematopoietic stem cell transplantation (SCT) (Razonable & Paya, 2003). After transplantation, an active hCMV infection occurs in approximately 60-70% of hCMV-seropositive patients or seronegative patients who receive organ transplants from a seropositive donor (Razonable & Paya, ibid). If no preventative measures are taken, the risk of developing hCMV disease is 20-30%. Considering the fact that approximately 26,000 allogenic SCTs were performed worldwide in 2008, the success of this therapy and the reduction of post-transplant morbidity and mortality have considerable financial implications. hCMV related complications can result in additional costs of EUR 25,000 to 50,000 per patient.
In addition, HCMV infection in transplant patients is associated with transplant related atherosclerosis and accelerated graft loss (Streblow et al., 2007).
As mentioned before, hCMV is relevant as a perinatal pathogen. Each year, approximately 1% of susceptible women seroconvert during pregnancy. Approximately 40% of these transmit hCMV to their children resulting in 40,000 infected newborns annually in the USA (Kenneson & Cannon, 2007). 10-20% of the infected children have acute symptoms at birth. Of these, up to 20% die and the remainder typically have moderate-to-severe complications, including CNS related conditions, like blindness, deafness and mental retardation. Apart from the devastating consequences for affected patients and their families, the healthcare costs for those patients are significant, in particular, if the perinatal infection results in severe and permanent disabilities.
To date, five antiviral agents are approved for use in hCMV infection: Ganciclovir/Valganciclovir, Cidofovir, Foscarnet and Fomivirsen. All compounds suffer from dose dependent side effects and the development of resistant virus strains (Schreiber et al., 2009). None of the drugs are licensed for use in children or in pregnant women. In addition, intravenous immunoglobulin preparations (IVIG) e.g. CYTOGAM® (Cytomegalovirus Immune Globulin Intravenous (Human), CSL Behring) and CYTOTECT® (Human immunoglobulin anti-cytomegalovirus, Biotest) are used for prophylaxis and treatment of patients at risk of hCMV infection. However, uncertainty about benefits of this treatment in the transplant situation is evident (Sokos et al., 2002; Raanani et al., 2009). The adoptive transfer of hCMV-specific cytotoxic T-cells has been used with success in hematopoetic SCT patients (Moss & Rickinson, 2005), but this treatment is extremely expensive and will be limited to a few transplant centres having the necessary expertise. Moreover, this type of treatment is restricted to transplant recipients who are seropositive for hCMV. In contrast, IVIG has been reported to be effective in the treatment and prevention of congenital CMV infection (Nigro et al., 2005). However, IVIG for hCMV treatment is isolated and purified from hCMV seropositive donors, resulting in variable titers and therefore batch-to-batch variation for hCMV specific antibodies in these preparations. In addition, human blood-derived drug products always bear the risk for transmission of human pathogens. As a consequence, a recombinant antibody product, allowing the efficient neutralisation of hCMV for the prophylaxis and treatment of diseases caused by hCMV infection is desired.
Targets for antibody therapy of hCMV infections are proteins expressed in the surface of the hCMV virion. The composition of the hCMV virion envelope is very complex and whilst many structural proteins that comprise the envelope have been identified, it is still not fully defined. During the development of antibodies for the therapy of hCMV, antigenic determinants have been identified in surface glycoprotein complexes gp58/116 (gB or gC-1), gp 47-52 (gC-II; gM and gN) (Shimamura et al., 2006) and gp 86 (gH or gC-III) (Urban et al., 1996). The majority of neutralising antibodies identified to date bind to gB protein, which has been shown to contain the majority of neutralising epitopes (Britt et al., 1990). The gB complex is synthesised as a 130 kD precursor, which is cleaved into two covalently linked molecules, named gp58 and gp116. The N-terminal fragment (gp116) contains one linear, neutralising epitope, called antigenic domain-2 (AD-2) of 20 amino acids (amino acids 67-86), which does not require complement for antibody-mediated biological activity (Meyer et al., 1990). The gp58 moiety of gB carries the neutralising domain AD-1, which may comprise 74 amino acids (amino acids 557-630) and most likely represents a conformational epitope (Ohlin et al., 1993; Wagner et al., 1992).
The advent of monoclonal antibodies initially gave rise to the identification of a variety of neutralising mouse monoclonal antibodies against hCMV. However, mouse monoclonal antibodies are unsuitable for use in human therapy since these proteins are recognised by the human immune system as being foreign, and are consequently eliminated after a very short period of time, resulting in low or no clinical efficacy. Chimeric antibodies have been developed against hCMV proteins and EP664834B (Harris et al) relates to a chimeric antibody targeted to the 86 kD glycoprotein of hCMV termed gH; however such antibodies have not been successful in clinical settings.
Technologies using heteromyelomas for the generation of hybridomas have been used to generate a variety of human monoclonal antibodies recognising various hCMV glycoproteins, which are found both in the viral envelope. U.S. Pat. No. 5,043,281 (Masuho et al) relates to a neutralising human monoclonal antibody that recognises a CMV antigen protein having a molecular weight of between 130,000 and 55,000. U.S. Pat. No. 5,750,106 (Ostberg) relates to a human monoclonal antibody to CMV termed SDZ MSL 109, which recognises the gH glycoprotein, as well as a hybridoma cell line for the production of this antibody. One of the virus-neutralising human monoclonal antibodies, SDZ MSL-109 has been evaluated in Phase I/II clinical trials for hCMV induced retinitis in immunocompromised patients, but due to lack of efficacy the clinical trials were not continued (Borucki et al., 2004; Hamilton et al., 1997; Boeckh et al., 2001). One plausible explanation for the failure of these trials is the antigenic variability of hCMV. hCMV is unique among the human herpes viruses in that it is antigenic variable and most human monoclonal antibodies, reacting with the envelope antigens, show strain-specific neutralisation capacity. This is especially true for the gH-specific human monoclonal antibodies, like SDZ MSL-109. This obstacle can only be overcome by the use of monoclonal antibodies directed against epitopes on hCMV that are conserved between different isolates.
In the past, progress in the isolation of hCMV neutralising monoclonal antibodies was slow, due to the fact that high-throughput screens of antibodies for neutralising capacity were not available. In addition, the method of Epstein Barr virus (EBV) immortalisation has been used frequently to generate immortalised B cells producing an antibody of interest, for a number of years. This technique has been successful for the generation of antibody-secreting cells from different sources of human B cells such as the peripheral blood of healthy subjects using antigen-specific selection (Casali et al., 1986), lymph nodes, spleen or peripheral blood from patients (Yamaguchi et al., 1987; Posner et al., 1991; Raff et al., 1988; Steenbakkers et al 1993 and 1994). This technique was used for the immortalisation of peripheral blood mononuclear cells isolated from CMV-seropositive blood donors and the subsequent isolation of three antibodies: ITC52, ITC63b and ITC88 (WO 93/021952 A1). ITC52 and ITC63b are reactive with the conformational AD-1 epitope of CMV consisting of amino acid sequence 557-630 of CMV gp58 and ITC88 is reactive against AD-2 comprising the amino acid sequence 67-86 (AD-2) of CMV gp116 (WO 93/021952 A1).
Improvements on the method of EBV transformation have been published by Lanzavecchia (WO 04/076677 A2) and Funaro et al (WO 07/068,758 A1) and these methods have been used for the generation of antibodies to hCMV. WO 08/084,410 A2 (Lanzavecchia & Macagno) relates to antibodies produced from EBV cell lines 1F11, 2F4, 5A2 and 9A11 that neutralise hCMV infection of endothelial cells, epithelial cells, retinal cells and dendritic cells and are directed towards a conformational epitope formed by gpUL130 and gpUL131A. However, the antibodies from these EBV lines do not have any detectable hCMV neutralising capacity, if fibroblasts are used as target cells for infection. WO 08/084,410 A2 also mentions EBV lines 1006, 5F1, 6B4 and 7H3, producing antibodies that neutralise hCMV infection of fibroblasts and endothelial cells at half-maximal inhibitory concentrations (IC50) ranging between 0.3 and 2.0 μg/ml. The antibodies produced from these EBV lines are described to bind to a functional epitope of gB. However, although antibody heavy and light chain sequences have been deducted from some of the above mentioned EBV cell lines, this data has not been confirmed for recombinantly expressed and purified antibodies encoded by the published sequences. A more recent patent application from Lanzavecchia & Macagno (WO 10/007,463 A1) relates to the antibody 6G4, which binds to an epitope determined by a combination of the UL128, UL130 and UL131A proteins and which neutralises hCMV infection of endothelial, retinal and dendritic cells. Furthermore, WO 10/007,533 A1 (Lanzavecchia & Macagno) relates to hCMV neutralising antibodies that bind to an epitope in the hCMV UL128 protein, an epitope formed by gH, gL, UL128 and UL130 proteins, an epitope formed by UL128, UL130 and UL131A proteins or an epitope formed by UL130 and UL131A proteins.
WO 08/071,806 A1 (Funaro et al) relates to the antibody 26A1, which binds to and neutralises hCMV but does not show significant binding to either antigens gB or gH when tested by ELISA. A half-maximal inhibitory concentration (IC50) of the antibody 26A1 is reported to be in the range of 1 μg/ml for both primary fibroblasts and endothelial cells, and therefore in a range that has been reached by antibodies described in the prior art. A further patent application by Funaro and colleagues, relates to the antibody 1F7, which recognises gH (WO 09/003,975 A1). Similar to antibody 26A1, as described in WO 08/071,806 A1, the half-maximal inhibitory concentration (IC50) of the antibody 1F7 is reported to be in the range of 1 μg/ml for both primary fibroblasts and endothelial cells, and therefore in a range that has been reached by antibodies described in the prior art. Yet another patent application by Funaro and colleagues (WO 09/024,445 A1) relates to the antibodies 8C10, 37B7, 8A11 and 10B7, which either recognise the AD-2 domain of gB (clones 8C10, 8A11, 10B7), or a protein unrelated to gB or gH (clone 37B7). As in the patent applications of Funaro and colleagues (WO 08/071,806 A1 and WO 09/003,975 A1), the antibodies described in WO 09/024,445 A1 also exhibit a half-maximal inhibitory concentration (IC50) in the range of 1 μg/ml for both primary fibroblasts and endothelial cells (10B7, 8A11, 37B7) or higher at about 10 μg/ml (8C10), and therefore in the range of previously published hCMV neutralising antibodies.
Additional recent patent applications, describe hCMV neutralising antibodies with similar features. For instance, WO 09/114,560 A2 (Olsen) relates to antibody clones 2F10, 2M16, 2N9, 3C21, 3G7, 4P12, 5P9, 9C16, which all bind to the AD-2 epitope of gB and display half-maximal inhibitory concentration (IC50) of hCMV infection of fibroblasts in the range of 1 μg/ml. US20090004198 (Nakajima et al) relates to a high affine antibody for the gB AD-1 domain, with apparent pM binding affinity, and 80% hCMV neutralising activity on fibroblasts, if used at concentrations of 1 μg/ml and higher (10 μm/ml and 100 μg/ml). Two recent applications WO 10/114,105 A1 and WO 10/114,106 A1, both from Evec Inc., describe antibodies that bind to AD-2 and a discontinuous epitope in AD-1, respectively.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.